1. Field of the Invention
The invention relates to modified Locked Nucleic Acid (LNA) units (e.g, individual LNA monomers and oligomers that include LNA monomers), particularly such monomers and oligomers having unique base groups. Desirable nucleobase and nucleosidic base substitutions can mediate universal hybridization when incorporated into nucleic acid strands. The novel LNA compounds may be used in a wide variety of applications, such as PCR primers, sequencing, synthesis of antisense oligonucleotides, diagnostics and the like.
2. Background
For disease states, classical therapeutics has generally focused upon interactions with proteins in an effort to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such protein is desired. By interfering with the production of proteins, the maximum therapeutic effect can be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would otherwise lead to the formation of undesired protein or proteins. One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence.
Oligonucleotides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other molecules. For example, the use of oligonucleotides as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies, and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides are also employed as primers in such PCR technology.
Oligonucleotides are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include i) generating synthetic labeled oligonucleotide probes for visualization after in situ hybridization, ii) generating microarray capture probes, iii) generating capture probes for nucleic acid sample preparations, iv) screening libraries with oligomeric compounds, v) DNA sequencing, vi) in vitro amplification of DNA by the polymerase chain reaction, vii) using fluorescently labeled oligonuclotides for real time vizualisation of PCR amplification efficiency (double dye probed, molecular beacons, and scorpions) and viii) site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also “DNA-protein interactions and The Polymerase Chain Reaction” in Vol. 2 of Current Protocols In Molecular Biology, supra. Oligonucleotides have even been used as building blocks in nanotechnology applications to make molecular structures with a defined geometry (cubes, cylinders etc.).
Certain chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase melting temperatures, Tm), to assist in identification of an oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
It would be desirable to have new nucleic acid compounds that could provide useful properties in a variety of application, including hybridization applications.